Atherosclerosis, a cardiovascular disease characterized by a thickening of the intima of muscular arteries, is the principal cause of myocardial and cerebral infarction, conditions which often ultimately result in death. The thickening occurs in large and mid-sized arteries, and may include fatty streaks, and later, in a markedly thickened layer that narrows the lumen of the vessel, resulting in clinical symptoms. The intimal thickening in advanced lesions or fibrous plaques includes a fibrotic layer of smooth muscle cells (SMC) and connective tissue matrix overlying a lipid-rich region beneath. The vessel becomes unstable under the stress of the high arterial flow rate as the lipid substructure is a weak supporting base. Additionally, accumulation of thrombotic material at the site of thickening may result in complete obstruction of the vessel lumen.
The etiology of atherosclerosis is unknown. However, it is hypothesized that an event which results in a change, injury, and/or disruption of the endothelial layer surrounding the lumen and overlying the SMC layer initiates the process. Upon sustaining the injury, a complex sequence of events is then initiated which leads to the ultimate formation of an atherosclerotic plaque. The endothelial cells becomes proliferative in an attempt to regenerate denuded regions of the lining of the vessel. Injury to endothelial linings is believed to cause circulating platelets to aggregate at the site of the injury where they adhere to exposed tissue at the wound site. Edema occurs at the site of injury, perhaps aiding the infiltration of macrophages which have migrated from blood and underlying tissue layers. These macrophages proliferate, and some ingest low density lipoproteins deposited at the site of injury, thereby becoming lipid-laden foam cells. The SMC at the site of the injury also change from a quiescent state to a synthetic state, proliferating and producing extracellular matrix materials such as collagen, elastin, and proteoglycans. A thickening extending into the lumen of the artery thus develops.
A high concentration of platelet-derived growth factor (PDGF) is found at the site of the lesion, and later, in the fibrous plaque (Barrett et al. (1988) Proc. Natl. Acad. Sci. 85:2810-2814). This growth factor is known to bind to receptors on the surface of various cells, thereby initiating a sequence of intracellular events that ultimately result in proliferation of those cells.
Native PDGF is a dimeric molecule consisting of two polypeptide chains, one or more of which appear to be glycosylated. The two chains (referred to as A or alpha and B or beta) are homologous but not identical. They have molecular weights of about 17,000 to 18,000 daltons and about 13,000 to 14,000 daltons, respectively. In vivo, the A and B chains are synthesized from larger precursors which are subsequently processed at the amino and carboxyl termini. The mature human A chain consists of 110 or 125 amino acids and various N-linked sugar side chains, the length and amino acid sequence being dependent on the tissue source. The fully processed human B chain is encoded by the C-sis gene and consists of 112 amino acids. It has been found to have a high degree of homology with the p28.sup.sis protein product of the v-sis transforming gene of simian sarcoma virus (SSV) (Johnsson et al., (1984) Embo. 3:921).
Biologically active PDGF can exist as an AA or BB homodimer, having a molecular weight of about 35,000 daltons (35 kD) or about 32 kD, respectively, or can take the form of an AB heterodimer having a molecular weight of about 34 kD. The human PDGF dimer is glycosylated and processed post-translationally into a three-dimensional conformation that is biologically active. This conformation is maintained by relatively weak noncovalent hydrogen bonds, hydrophobic and charge interactions, and strong covalent bonds between sulfur atoms in cysteine residues. The PDGF dimer has eight such disulfide linkages which exist both between chains (interchain bonds) and within the same chain (intrachain bonds). Reduction of either the AA or BB dimer into its component monomeric chains destroys all biological activity.
Different cell types are known to elicit different dimeric forms of PDGF. In fact, many of the cells intimately involved in the formation of the plaque produce and secrete various forms of PDGF. For example, platelets aggregating at the site of initial injury at the endothelial lining release PDGF AB. Macrophages produce PDGF BB, and SMC and endothelial cells produce PDGF AA.
Platelet-derived growth factor has been postulated to be the etiological agent in atherosclerosis (see e.g., Rutherford et al. (1976) J. Cell. Biol. 69:196-203; Friedman et al. (1977) J. Clin. Invest. 60:1191-1201). The released PDGF is able to chemotactically recruit fibroblasts, monocytes, glia, and smooth muscle cells to migrate to the site of the wound. The released PDGF also acts as a mitogen by stimulating DNA synthesis in these cells, thereby increasing their proliferation rate. Quiescent SMC normally found in nonembryonic arterial walls, becomes synthetic and proliferative upon stimulation with the PDGF produced by endothelial cells, macrophages, and platelets. In this active state, SMC, themselves, produce PDGF AA which in turn, activates quiescent SMC.
It has been hypothesized that inhibiting the activity of PDGF may inhibit or reverse the formation of atherosclerotic plaques. To that end, a number of different molecules were tested as inhibitors or antagonists of PDGF. For example, fenofibrate (Kloer (1987) Am. J. Med. 83(B):3-8) and retinoic acid (Mordan (1989) Cancer Res. 49:906-909) inhibit PDGF-dependent stimulation of DNA synthesis. Monoclonal antibody C3.1 (Kawahara et al. (1987) Biochem. Biophys. Res. Commun. 147:839-845) and 5-methyl-7-diethylamino-s-triazolo (I,5-a) pyrimidine (Ohnishi et al. (1983) Life Sci. 31:2595-2602; Tiell et al. (1983) Artery 12:33-50) are PDGF antagonists. Interferon inhibits PDGF-induced protein synthesis in fibroblasts (Zagari et al. (1988) Biochem. Biophys. Res. Commun. 150:1207-1212) and inhibits the mitogenic effect of PDGF on fibroblasts (Hosang (1988) J. Cell. Physiol. 194:396-404). Suramin binds to PDGF and inhibits its biological activity (Hosang (1985) J. Cell. Biochem. 29:265-273), and protamine inhibits the binding of PDGF to its receptor (Huang (1984) J. Cell. Biol. 26:205-220).
The object of this invention is to inhibit the binding of PDGF to its receptors on responsive cells, and thus to inhibit the subsequent biological activities triggered by the binding of active PDGF to its receptors. It is also an object of the present invention to inhibit the formation of atherosclerotic lesions and fibrous plaques by inhibiting the biological activity of PDGF. Another object is to stop and/or to reverse the progression of atherosclerosis. Another object is to inhibit the proliferation of smooth muscle cells at the site of arterial injury or insult. Yet another object is to prevent the migration and proliferation of macrophages within the sub-intimal endothelial layer of mid- and large-sized muscular arteries.